Renal secretory transport of organic anions (OA) and organic cations (OC) controls the excretion of most foreign chemicals and/or their metabolites. Our research focus is the biochemistry of these transport proteins; their development, expression, and control; and their impact on the toxicity of xenobiotics. We have previously characterized the mechanisms and energetics of both processes and cloned several of these transport proteins using expression cloning of a rat kidney library in Xenopus oocytes. We were the first to clone the basolateral transporter mediating OA/alpha-ketoglutarate exchange, rOAT1, the critical uphill step in elimination of negatively charged xenobiotics and their metabolites. Homology screening yielded the human homolog. Function of the homologs was identical in mechanism, but the human form had a 5-10- fold higher affinity for all substrates tested. In particular, its Km for the nucleoside phosphonate antiviral drugs, adefovir and cidofovir, were 30-50 uM, indicating that hOAT1 mediated cellular accumulation was the probable basis for their nephrotoxicity. The human form has been stably transfected into MDCK cells, a polarized renal cell line. This model will be used to characterize its function and cellular localization in a mammalian system. We have also generated a cDNA construct in which green fluorescent protein (GFP) is linked in frame with rOAT1. When transfected into oocytes, cell lines, or isolated tubules, this construct produces a functional, fluorescent protein which can be followed optically in the living cell. These studies have confirmed the basolateral targeting of rOAT1, as predicted based on its physiology. We have also cloned an OC transporter (rOCT2), and shown that it mediates potential driven entry of OC in oocytes and stably transfected MDCK cells. In addition, an GFP construct was generated for OCT2. The resulting protein, rOCT2-GFP, was shown to be functionally active and to be localized exclusively to the basolateral face of the tubule. Thus, both functional and localization data indicate that rOCT2, contrary to the hypothesis of others, mediates potential-driven basolateral uptake of OC, not apical proton/OC exchange. Finally, rOCT2 was stably transfected into NIH3T3 cells and shown to be as easily studied model for the evaluation of transport and toxicity of cationic drugs. - renal, oganic anions, organic cations, transport, Xenopus oocytes, cloning